The effect of different temperatures on the Scatchard analysis of binding studies with 125I-hCG and intact rat testis Leydig cells has been investigated. The results show that at temperatures greater than 4°C an overestimation of the number of receptors/cell is likely to occur. A one-site analysis of the data gives values of 13573, 61924 and 3802 LH receptors/cell at 34°C., 21°C and 4°C respectively. At 34°C and 21°C (but not at 4°C) a two-site analysis of the data is possible, giving similar high affinity binding components at both temperatures ( K D ∗ 1 × 10 −10 M ) but with dissimilar low affinity binding components (34°C, 2.17 ×X 10~9 M and 21°C., 6.3 X 10~8 M). The calculated total number of LH receptors using a two-site analysis at 21 °C is equal to 114766 receptors/cell, and at 34 °C it is 37987 receptors/cell. It is proposed that the differences in the level of binding at different temperatures and the associated changes in the value of K D, reflect the temperature sensitivity of the endocytic pathway of th 123e 125I-hCG/LH receptor complex within the rat testis Leydig cell. From previous studies it is known that at 34 °C and 21°C., a large part of the cell-associated hormone is not receptor bound but rather it represents ‘processed’ hormone before it is released from the cell. At 4°C the cell-associated 125I-hCG remains bound only to cell surface LH receptors and thus gives a more accurate measure of total receptor numbers/cell.